Suidatrestin and production thereof

ABSTRACT

A novel trehalase inhibitor having the following physicochemical properties: 
     (1) Activity: a specific inhibitory activity to trehalase; 
     (2) Form: white powder, water-soluble; 
     (3) Somogy-Nelson test: negative 
     (4) Rydon-Smith test: positive 
     (5) Optical rotation [α] D   23  : 113.8° (C, 0.1, H 2  O) 
     (6) Molecular weight: 335 ([M+H] +  =336) 
     (7) Ultraviolet absorption spectrum: 258.5 nm (max.) 
     (8)  13  C-NMR(ppm): 28.9, 41.0, 56.9, 58.4, 64.3, 65.0, 71.3, 74.0, 75.2, 75.8, 76.0, 77.1, 123.0, 144.7

This is a divisional of application Ser. No. 08/040,184, filed on Mar.29, 1993, now U.S. Pat. No. 5,354,685.

FIELD OF THE INVENTION

The invention relates to a novel trehalase inhibitor. The inventionfurther relates to an actinomycete capable of producing Suidatrestin andto a method for the production of Suidatrestin by using theactinomycete.

PRIOR ART

Trehalase is an enzyme which decomposes trehalose into its unitmonosaccharide, glucose. It is known that said enzyme is present notonly in small intestine and kidney of mammals but also in fungi andinsects in which trehalose is a main source of energy for livingactivity. Trehalase is therefore an important enzyme which plays a mainpart in energy-metabolism in fungi and insects. In contrast, there isobserved no useful role of the enzyme in kidney and small intestine ofmammals. Recently, Validmaycin which specifically inhibit the trehalaseof Rhizoctonia solani bacter was found, and it has been used as anantifungal agent. Furthermore we found the inhibitor, Trehalostatin,which has a specific effect to the trehalases of insects and we alsohave been studied its use as an antifungal agent and insecticide.However, it is anticipated that there will be an appearance of fungi andinsects which are resistant to the above agents. Accordingly, it hasbeen desired to develop a new trehalase inhibitor which has a differentstructure and inhibitory characteristics from the prior trehalaseinhibitors.

SUMMARY OF THE INVENTION

It was considered that a trehalase inhibitor which specifically inhibitsthe mammalian trehalases might inhibit trehalases having resistance tothe conventional trehalase inhibitors. Additionally, because trehalasesdo not have an important role in mammals, it can be expected that thetrehalase inhibitor has a minimum side effect to mammals.

Accordingly, the present inventors had searched widely in nature for amicroorganism which can produce a substance capable of stronglyinhibiting a trehalase in porcine small intestine. The present inventorshad found that actinomycete belonged to a genus of Streptomyces, whichwhen isolated from soil, produced a new substance which showed aninhibitory effect to trehalase of animals especially mammalian smallintestine with a very low concentration, in both their culture mediumand mycobiont. The new substance had been named as "Suidatrestin". Theactinomycete according to the present invention is preferably a strainSAM 1953 isolated from soil. The SAM 1953 strain has the followingbacteriological properties:

(1) Morphological characteristics

The substrate and aerial mycelium was 0.4-0.5 μm in width and welldiverged. The substrate mycelium was branched. Fragmentation of thesubstrate mycelium was not observed. The aerial mycelium is alsobranched and formed spore chains with more than 10 spores per chain. Thespore chains formed loops, hooks, and sometimes imperfect spirals. Thespores were spherical to cylindrical in shape and 0.8 to 1.0 μm indiameter. The spore surface was smooth. No sporangia, synnemate, orselerotia were observed even after 14 days cultivation.

(2) Cultural characteristics (28° C., 14 days culture)

Sucrose.nitrate agar medium; growth: poor; aerial hypha: slight, lightbrown; reverse side of colony: light brown; soluble pigment: none

Glucose.asparagine agar medium; growth: good; aerial hypha: abundant,yellow; reverse side of colony: dark orange; soluble pigment: none

Glycerine.asparagine agar medium; growth: good; aerial hypha: abundant;ashy brown; reverse side of colony: ashy brown; soluble pigment: none

Starch.inorganic salt agar medium; growth: good; aerial hypha: abundant,dark green; reverse side of colony: ashy brown; soluble pigment: none

Tyrosine agar medium; growth: good; aerial hypha: abundant, brownishash; reverse side of colony: reddish brown; soluble pigment: none

Nutrient agar medium; growth: good; aerial hypha: abundant, white;reverse side of colony: white; soluble pigment: none

Yeast-extraction.malt-extraction agar medium; growth: good, aerialhypha: abundant, greeny ash; reverse side of colony: dark brown; solublepigment: yellowish brown

Oat meal agar medium; growth: good, aerial hypha: abundant, brownishash; reverse side of colony: yellow; pigment: none

Tap water agar medium; growth: poor, aerial hypha: poor, reverse side ofcolony: semitransparency; soluble pigment: none

1/10 carrot.potato agar medium; growth: good; aerial hypha: abundant,brown; reverse side of colony: brown; soluble pigment: none

1/10 V-8 juice agar medium; growth: good; aerial hypha: abundant, ashybrown; reverse side of colony: brown; soluble pigment: none

(3) Physiological characteristics

1) Growth temperature

As a result of culture tests conducted at temperature of 12.5° C., 15.5°C., 18.0° C., 21.0° C., 23.5° C., 26.0° C., 28.5° C., 30.5° C., 33.0°C., 35.5° C., 38.5° C. and 41.0° C. by using a trypton.yeast extractliquid medium, the temperature range for growth was 12.5° to 41.0° C.,with optimum growth occurring 21.0°-38.5° C.

    ______________________________________    2)    Liquefaction of gelatin          glucose.peptone.gelatin medium                                   positive          simple gelatin medium    positive          meat extract.gelatin medium                                   positive    3)    Hydrolysis of starch     positive    4)    Coagulation of skim milk (28° C.)                                   negative    5)    Peptonization of skim milk                                   positive    6)    Formation of melanoid pigment          peptone.yeast-extract iron agar medium                                   negative          tyrosine agar medium     negative          tryptone yeast-extract agar medium                                   negative    7)    Reduction of nitrate     negative    8)    Utilization of Carbohydrates of carbon source          (determined in the basal medium of Pridham and Gott-          lieb (ISP-9) at 28° C. for 14 days).            D-glucose                    +            D-xylose                    +            L-rhamnose                    -            L-arabinose                    +            D-fructose                    +            Raffinose                    -            D-mannitol                    +            Inositol                    -            Sucrose -            Lactose +            Salicin ±    ______________________________________     (+; utilized, ±; doubtful to use, -; not utilized)

(4) Chemotaxonomy

1) 2,6-diaminopimelic acid

By investigations of the whole body of the strain according to themethod of Staneck, J. L. and Roberts, G. D. method (Applied MicrobiologyVol. 28, P. 226, 1974), there was observed LL-2,6-diaminopimelic acid.

2) Menaquinones

The principal menaquinone component is MK-9 (Hb, H8).

According to those taxonomic properties, the SAM 1953 strain wasidentified as an actinomycete belonging to the genus Streptmyces. Thus,this present invention can use any strain that belongs to the genusStreptomyces and provides Suidatrestin. The strain SAM 1953 was named asStreptomyces sp. SAM 1953, and was deposited with Fermentation ResearchInstitute Agency of Industrial Science and Technology, Ministry ofInternational Trade and Industry 1-3, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, 305, Japan, on Mar. 25, 1992 (Accession No. FERM BP-3805).

In present invention, the above actinomycete may be cultured with eitherliquid or solid medium. However, it is usually advantageous to carry outa shaking culture with the liquid medium or aeration-agitation culture.Any medium may be used, in which the actinomycete according to thepresent invention can grow and the product of the present invention canbe accumulated. Namely, there may be used as carbon source glucose,lactose, starch, sucrose, dextrin, molasses, organic acids etc. andthere may be used as nitrogen source protein hydrolysate such as peptonecasamino acid, and also meat-extact, yeast-extract, soybean cake, cornsteep liquor, amino acid, ammonium salt, nitrate, and other organic orinorganic nitrogen compounds.

Further, it may be added as an inorganic salt many types of phosphate,magnesium sulfate, sodium chloride, and the like, as well as vitamins,compound bearing nucleic acid -associated compounds in order to promotethe growth of the strain. Sometimes, it is effective to add anantifoaming agent such as silicon, polypropylene-glycol derivatives andsoybean oil into the medium for increasing an amount of accumulation ofthe novel substance according to the present invention.

It is preferable to carry out a small-scale preculture and to inoculatethe culture product into a main culture medium. In both the main cultureand preculture, the culture conditions such as temperature, period,medium properties may be selected and regulated so as to obtain amaximum amount of accumulation of the novel substance of this invention,but it is usually preferable to maintain the culture under aerobiccondition at a temperature of from 25° C. to 40° C. The pH value of aliquid medium is preferably kept in a range of from 5.5 and 8.0.

When the liquid medium is used for culture, a desired product is mainlyaccumulated in a liquid part. It is therefore desired to remove cells byfiltration or centrifugation and separate them from supernatant offiltrate. It is also possible, if desired, to obtain the desired productdirectly from the culture medium.

The inhibitory activity of Suidatrestin in each step during purificationis determined by the following method.

Inhibitor solution (50 μl) and trehalase solution (porcine entrails) 50μl are mixed, and incubated at 37° C. for 5 min. After addition of 400μl of 5 mM phosphate buffer (pH 6.3) containing 5 mM trehalose, theresulting mixture is incubated at 37° C. for 15 min. and stopped byadding 1N hydrochloric acid (30 μl). Then, into these 100 ul reactantsolution, there are added 0.1M phosphate buffer (pH 6.5) 2.5 ml, 4 mMABTS 25 μl, 3.5-diaminobenzoic acid 25 μl, and horseradish-peroxidaseaqueous solution (1 mg/ml) 10 μl. The resulting mixture is incubated at37° C. for 5 min., then mixed with glucose-oxidase solution (1 mg/ml)100 μl and further incubated at 37° C. for 10 min., followed by additionof 1M sodium azide aqueous solution (100 μl) to measure absorbance at550 nm and obtain an amount of a produced glucose.

In this method, a concentration of Suidatrestin which inhibits activityof the enzyme(trehalase) by 50% is estimated as one unit/ml.

The present substance may be separated and purified by various methodsbased on the chemical characteristics of the same product.

The Suidatrestin may be isolated as a white and amorphous powder by, forexample, treatment with an organic solvent, gel-filtration with cephadexand biogel, ion-exchange chromatography with various ion-exchangeresins, adsorption chromatography with adsorbent such as Amberlite-XAD-1and XAD-2, active carbon, silicagel, normal-phase chromatography with acarrier such as YMC-PA43 and TSK-Amide 80, or optionally combinationsthereof. Other methods may be used so long as the properties of thepresent substance are effectively utilized.

An especially preferred combination of the absorbents are that of Dawex50W-X2 (H⁺), active carbon, YMC-PA43 and TSK-Amide 80.

The novel substance thus obtained has properties shown in the followingexamples. It shows the inhibitory activity to trehalase of fungi,insects, especially of porcine small intestine under a very lowconcentration as shown in the examples. It is therefore very useful asfungicide, insecticides, or anti-corpulency diabetes agent.

Further, the present substance can be used not only in an isolated formbut also as the culture product per se of Suidatrestin-producing cellsor simply purified one therefrom.

The present invention will be explained in detail with reference to thefollowing examples which should not be construed as limiting the scopeof the invention.

EXAMPLE Example 1:

Production of Suidatrestin

Pure culture of SAM 1953 strain 60 ml was inoculated in the medium (pH7.0, 6 l) containing glucose, yeast-extract, peptone, and magnesiumsulfate, and subjected to aeration-agitation culture for 120 hrs inthree 10 l jar-fermenters (33° C., 200 rpm, 1 vvm). Cells were removedfrom these culture fluid by filtration using filterpaper (TOYO-ROSHI,No. 2). The resulting filtrate was passed through a column (Dowex 50W-X2(H⁺), 3.5 l) equilibrated with deionized water so as to adsorb a portionof trehalase-inhibiting activity.

The column was washed with deionized water of 6-column volume and theportion of trehalase-inhibiting activity was eluted with 0.1N NH₄ OH of16-column volume. The eluate was fractioned by 500 ml to collectfractions of trehalase-inhibiting activity.

Ammonia was removed from the fractions of trehalase-inhibiting activityby using an evaporator to a final volume of 300 ml. A part of theresulting concentrate was loaded onto CK08P (Na⁺) column (2.5 φ×20 cm)equilibrated with 20 mM sodium phosphate buffer so as to adsorbtrehalase-inhibiting activity. The column was washed with 500 ml of thesame buffer and trehalase-inhibiting activity was eluted out with alinear-gradient consisting of 500 ml each of 20 mM sodium phosphatebuffer (pH 6.0) and 0.2M sodium phosphate buffer (pH 8.8). The sameoperations were repeated on the other concentrates.

The trehalase-inhibiting activity fractions thus obtained were adsorbedto an active carbon column (2.5 φ×10 cm) washed with sufficient water.The column was washed with 700 ml of deionized water and thetrehalase-inhibiting activity portion was eluted with 500 ml of 30%MeOH.

The trehalase-inhibiting activity fractions thus obtained wereconcentrated to 5 ml by an evaporator and the concentrate was subjectedto re-chromatography with CK08P (Na⁺) column (2.5 φ×20 cm). Thefractions of trehalase-inhibiting activity thus obtained wereconcentrated to 5 ml by using an evaporator, injected into a Carbonexcolumn washed with distilled water, washed with distilled water, andeluted with 0-30% MeOH gradient.

The fractions of trehalase-inhibiting activity thus obtained wereconcentrated to 2 ml by using an evaporator, injected into YMC PA43column equilibrated with acetonitrile:distilled water=75:25 (v/v) anddeveloped with the same solvent.

The fractions of trehalase-inhibiting activity thus obtained wereconcentrated to 2 ml by using an evaporator, injected into TSK-Amide 80column (1.0 φ×45 cm) equibrated with acetonitrile:distilled water=63:35(v/v) containing 20 mM sodium phosphate beffer (pH 5) and developed withthe same solvent to monitor the absorbance of eluate on 210 nm. In thischromatography, trehalase-inhibiting activity was eluted as a singlepeak showing absorbance on 210 nm.

The fractions of trehalase-inhibiting activity thus obtained wereconcentrated to 2 ml by using an evaporator and subjected tore-chromatography with Carbonex column.

The fractions of trehalase-inhibiting activity thus obtained wereconcentrated to 2 ml by using an evaporator, injected into AsahipakNH2P-50 column equilibrated with acetonitrile:distilled water=85:15(v/v), and developed with the same solvent. By concentrating, drying andlyophilizing trehalase-inhibiting activity fractions with diluted HCl,the desired product was obtained in a pure form (1 mg).

Example 2:

Physicochemical properties of Suidatresin

The substance obtained from the culture of SAM 1953 strain according toExample 1 was named Suidatrestin.

Physicochemical properties were illustrated below.

Form: white powder; water-soluble

Somogy-Nelson test: negative

Rydon-Smith test: positive

Optical rotation [α]_(D) ²³ :113.8° (C, 0.1, H₂ O)

Molecular weight: 335 ([M+H]⁺ =336)

Ultraviolet absorption spectrum: 258.5 nm (max.)

¹³ C-NMR (ppm): 28.9, 41.0, 56,9, 58.4, 64.3, 65.0, 71.3, 74.0, 75.2,75.8, 76.0, 77.1, 123.0, 144.7

Example 3:

The enzyme inhibiting spectrum of Suidatrestin

The trehalase-inhibiting activity of Suidatrestin was measured againstvarious trehalases.

    ______________________________________    Origin of trehalase                      ID.sub.50 (ng)    ______________________________________    porcine small intestine                      0.21    Aldrichina grahami                      1.05    fungi             0.93    ______________________________________

What is claimed:
 1. Isolated and purified trehalase inhibitor which hasthe following physicochemical properties:(1) Activity: a specificinhibitory activity to trehalase; (2) Form: water-soluble white powder;(3) Somogy-Nelson test: negative; (4) Rydon-Smith test: positive; (5)Optical rotation [α]_(D) ²³ : 113.8° (C, 0.1, H₂ O); (6) Molecularweight: 335 ([M+H]⁺ =336) (7) Ultraviolet absorption spectrum: 258.5 nm(max.) (8) ¹³ C-NMR(ppm): 28.9, 41.0, 56.9, 58.4, 64.3, 65.0, 71.3,74.0, 75.2, 75.8, 76.0, 77.1, 123.0, 144.7.
 2. A method for theproduction of the trehalase inhibitor according to claim 1, comprisingculturing Streptomyces genus actinomycete which can produce thetrehalase inhibitor to obtain an actinomycete culture containingtrehalase inhibitor and purifying the trehalase inhibitor by treatingthe resulting culture using one or more of the following processsteps:(1) by loading the actinomycete culture on an ion-exchangechromatographic column and eluting it with a solution containing anincreasing pH or ionic strength to obtain an eluate containing thetrehalase inhibitor, (2) adsorbing an eluate containing the trehalaseinhibitor on an active carbon column and eluting it with alcohol, or (3)loading an eluate containing the trehalase inhibitor on a normal phasecolumn chromatography and eluting it with an equilibration solvent.
 3. Amethod for the production of the trehalase inhibitor according to claim1, comprising culturing Streptomyces genus actinomycete which canproduce the trehalase inhibitor to obtain an actinomycete culturecontaining trehalase inhibitor and purifying the trehalase inhibitor bytreating the resulting culture by loading the actinomycete culture on anion-exchange chromatographic column and eluting it with a solutioncontaining an increasing pH or ionic strength to obtain an eluatecontaining the trehalase inhibitor.
 4. A method for the production ofthe trehalase inhibitor according to claim 1, comprising culturingStreptomyces genus actinomycete which can produce the trehalaseinhibitor to obtain an actinomycete culture containing trehalaseinhibitor and purifying the trehalase inhibitor by treating theresulting culture by adsorbing a solution containing the trehalaseinhibitor on an active carbon column and eluting it with alcohol.
 5. Amethod for the production of the trehalase inhibitor according to claim1, comprising culturing Streptomyces genus actinomycete which canproduce the trehalase inhibitor to obtain an actinomycete culturecontaining trehalase inhibitor and purifying the trehalase inhibitor bytreating the resulting culture by loading a solution containing thetrehalase inhibitor on a normal phase column chromatography and elutingit with an equilibration solvent.